Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Immunoblots showing immunoreactivity of GATA4 and GATA6 proteins with preabsorbed rabbit anti-GATA4 (sc-9053) and anti-GATA6 (sc-9055) antibodies. A, Demonstration of specificity of preabsorbed GATA4 and GATA6 antibodies in detecting their respective recombinant porcine proteins. In vitro translated recombinant (rec) GATA4 and GATA6 (or vector only) were used for immunoblots and probed with preabsorbed GATA4 (upper image) and GATA6 (lower image) antibodies. B, Examples of immunoblots from granulosa cell RNAi studies probed with preabsorbed GATA4 (upper image) and GATA6 (lower image) antibodies. ns, indicates a non-specific band from the same blot and is included to show relative loading.
• Supplemental Figure 2 - Caspase-3 dependent apoptosis is not detected in granulosa cells under RNAi knockdown conditions. Following an initial attachment period, porcine primary granulosa cell cultures were transfected with control (C), GATA4 (G4), GATA6 (G6), or combined GATA4 + GATA6 (G4+G6) RNAi molecules for 24 h then allowed to recover for 72 h prior to treatment with 8-Bromo-cAMP (8-Br-cAMP, 1 mM) or vehicle for 6 and 24 h. Cytoplasmic protein was subjected to immunoblot with a caspase-3 antibody which detects both the inactive and active forms of caspase-3. The positive control lane represents granulosa cells treated with H-89 cultured under conditions previously described to activate caspase-3 (26). The 17 kDa active fragment of caspase-3 was not detected in granulosa cells under any RNAi conditions. Blots are representative of 3 different experiments.